Abstract
The C-X-C chemokine receptor 4 (CXCR4) is G protein-coupled receptor that upon binding to its cognate ligand, can lead to tumor progression. Several CXCR4-targeted therapies are currently under investigation, and with it comes the need for imaging agents capable of accurate depiction of CXCR4 for therapeutic stratification and monitoring. PET agents enjoy the most success, but more cost-effective and radiation-free approaches such as ultrasound (US) imaging could represent an attractive alternative. In this work, we developed a targeted microbubble (MB) for imaging of vascular CXCR4 expression in cancer. A CXCR4-targeted MB was developed through incorporation of the T140 peptide into the MB shell. Binding properties of the T140-MB and control, non-targeted MB (NT-MB) were evaluated in MDA-MB-231 cells where CXCR4 expression was knocked-down (via shRNA) through optical imaging, and in the lymphoma tumor models U2932 and SuDHL8 (high and low CXCR4 expression, respectively) by US imaging. PET imaging of [18F]MCFB, a tumor-penetrating CXCR4-targeted small molecule, was used to provide whole-tumor CXCR4 readouts. CXCR4 expression and microvessel density were performed by immunohistochemistry analysis and western blot. T140-MB were formed with similar properties to NT-MB and accumulated sensitively and specifically in cells according to their CXCR4 expression. In NOD SCID mice, T140-MB persisted longer in tumors than NT-MB, indicative of target interaction, but showed no difference between U2932 and SuDHL8. In contrast, PET imaging with [18F]MCFB showed a marked difference in tumor uptake at 40–60 min post-injection between the two tumor models (p<0.05). Ex vivo analysis revealed that the large differences in CXCR4 expression between the two models are not reflected in the vascular compartment, where the MB are restricted; in fact, microvessel density and CXCR4 expression in the vasculature was comparable between U2932 and SuDHL8 tumors. In conclusion, we successfully developed a T140-MB that can be used for imaging CXCR4 expression in the tumor vasculature.
Highlights
The C-X-C chemokine receptor 4 (CXCR4, known as fusin and CD184) is a seven-transmembrane domain G protein-coupled receptor (GPCR)
These pathways are exploited during tumorigenesis, especially in the metastatic process, where CXCR4-expressing tumor cells are chemotactically homed to organs with abundant levels of CXCL12—such as the liver, bone marrow, lungs and lymph nodes [2,3,4]
Whereas non-targeted MB (NT-MB)’ shell is composed of the lipids DPPC, DPPA and DSPE-PEG2000-NH2, the targeted MB (T140-MB) were developed by replacing half (4.8%) of the original mole fraction of DSPE-PEG2000- NH2 in the MB’ shell with the lipo-PEG-peptide, DSPE-PEG2000-T140 (S1 and S2 Tables). We investigated whether this modification of the MB’ shell would introduce variability in certain properties such as size and concentration; T140-MB and NT-MB were produced with similar concentrations (9x108 MB/ml and 1x109 MB/ml, respectively—Fig 1B) and size distribution (1.7±0.7 and 2.0±0.9 μm, respectively—Fig 1C), as determined by counting and sizing analysis of the FOVs acquired (Fig 1D), with small oscillations throughout the 3 weeks evaluated
Summary
The C-X-C chemokine receptor 4 (CXCR4, known as fusin and CD184) is a seven-transmembrane domain G protein-coupled receptor (GPCR). Upon binding its natural ligand, the CXCL12 chemokine ( known as stromal cell-derived factor-1α, SDF-1α), CXCR4 activates several downstream signaling pathways leading to altered gene expression, migration, survival and proliferation [1]. These pathways are exploited during tumorigenesis, especially in the metastatic process, where CXCR4-expressing tumor cells are chemotactically homed to organs with abundant levels of CXCL12—such as the liver, bone marrow, lungs and lymph nodes [2,3,4]. Despite the relative success of these PET agents, their clinical use is limited by important considerations such as radiation dose, high cost, and relative inaccessibility of PET scanners
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